Treatment

ABSTRACT

The present invention provides a specific binding molecule which binds to Annexin-1 (Anx-A1) for use in the treatment of T cell-mediated disease.

The present invention relates to methods for treating T cell-mediateddisease by modulating the activity of Annexin-1.

Autoimmune diseases are chronic disabling pathologies caused bymalfunction of the immune system. In most cases they are initiated by anuncontrolled T cell response to autoantigens presented in the context ofMHC molecules of antigen presenting cells (APCs). Several factors havebeen described as being involved in the pathogenesis of autoimmunediseases including environmental, genetic and viral factors, with oneoverarching feature: the hyperresponsivity of T cells.

Glucocorticoids (GCs) are often used for the therapy of a variety ofchronic autoimmune diseases because of their ability to simultaneouslyblock both the innate and adaptive immune response. Studies over thelast 10 years or so by the present inventors and other research groupshave shown that some of the inflammatory effects of GCs on the innateimmune response are mediated by a protein called Annexin-1 (Anx-A1).This protein has been proven to exert a homeostatic control over anumber of cell types including neutrophils, macrophages and endothelialcells. However, one aspect that has always been neglected is the role ofAnx-A1 in the adaptive immune response. This is surprising consideringthat Anx-A1 has been proposed as one of the second messengers of thepharmacological effects of GCs.

The present inventors have previously shown that Anx-A1 plays ahomeostatic role in T cells by modulating the strength of T cellreceptor (TCR) signaling (D′Acquisto et al., Blood 109: 1095-1102,2007).

Furthermore, the inventors have shown that high levels of Anx-A1 lowerthe threshold of T cell activation and favour the differentiation intoTh1 cells, whereas Anx-A1 deficient mice show impaired T cell activationand increased differentiation into Th2 cells (D′Acquisto et al., Eur. J.Immunol. 37: 3131-3142, 2007).

WO 2005/027965 describes the discovery of a mechanism by which apoptoticneutrophils deliver anti-inflammatory signals to dendritic cells andidentifies an antibody that interferes with this process. In particular,WO 2005/027965 describes the identification of Anx-1 as a signallingmolecule which is said to be expressed by apoptotic neutrophils toinhibit the activation and maturation of dendritic cells. WO 2005/027965proposes that an antibody termed DAC5 (Detector of Apoptotic Cells Nr.5) recognizes and blocks the anti-inflammatory effects of Anx-1presented on the surface of apoptotic neutrophils upon phagocytosis bydendritic cells. WO 2005/027965 thus refers to the possibility oftreatment of various diseases by targeting such apoptotic cells anddeleting them by causing an inflammatory response, but does not discussa role for Anx-1 in T cell activation.

WO 2005/027965 claims that annexins are expressed on cells that areundergoing apoptosis (see for example page 8, lines 6-7 and 29-30) andthat these annexins are presented on the surface of such cells (see forexample page 6, lines 10-11 and page 8, lines 16-17). However, twoseparate studies (Maderna et al., J Immunol., 174: 3727-3733, 2005;Scannell et al., J Immunol., 178: 4595-4605, 2007) have shown thatapoptotic cells, including neutrophils, release annexin-1, rather thanexpressing the protein and presenting it on the cell surface. Sinceannexin-1 is released from the cell, it cannot be claimed that DAC5would identify only apoptotic cells expressing the protein on thesurface, as the antibody would also identify released annexin-1.

Furthermore, WO 2005/027965 claims that co-incubation of apoptoticneutrophils expressing annexin-1 on their cell membrane with dendriticcells activated with LPS causes inhibition of TNF-α secretion andupregulation of the activation markers CD83, CD86 and HLA-DR, and thataddition of DAC5 to this culture reverses the inhibitory effects of theannexin-1 expressing apoptotic neutrophils (page 5, line 31 to page 6,line 8). Data from the present inventors (Huggins et al., FASEB J. 2008,in press) demonstrates that dendritic cells release Anx-1 uponstimulation with LPS and thus the DAC5 described in WO 2005/027965 wouldbind the Anx-1 externalized on the neutrophils as well as the annexin-1released by dendritic cells. Furthermore, the present inventors havefound that the absence of annexin-1 in dendritic cells causes anincreased expression of maturation/activation markers and production ofinflammatory cytokines such as TNF-cc and IL-113 and IL-12. Therefore,the antibody DACS described in WO 2005/027965 should affect thematuration and activation of dendritic cells and thus the subsequentmodulation of the immune response.

In support of this, the present inventors have shown that co-culturingAnx-A1^(−/−) dendritic cells with nave T cells within a mixed lymphocytereaction (MLR) showed a significantly reduced ability to induce either Tcell proliferation or IL-2 and IFN-γ production. Thus, agents blockingAnx-A1 function in dendritic cells should reduce their capacity tostimulate a robust T cell mediated immune response. The antibodiesreferred to in WO 2005/027965 would therefore not be suitable fortreating the diseases referred to in that patent application.

The present invention provides the use of specific binding moleculeswhich bind to Annexin-1 (Anx-A1) in the treatment of T cell-mediateddisease.

According to a first aspect of the invention there is therefore provideda specific binding molecule which binds to Annexin-1 (Anx-A1) for use inthe treatment of T cell-mediated disease.

The present inventors have previously shown that Anx-A1 modulates thestrength of T cell receptor (TCR) signaling and that high levels ofAnx-A1 lower the threshold of T cell activation and favourdifferentiation into Th1 cells. The inventors have now identified theannexin pathway, and the ensuing signal, as a target for blockade inorder to treat T cell-mediated diseases. Such diseases include those inwhich there is aberrant T cell activation, for example many autoimmunediseases, and those in which it is desirable to skew differentiation ofT cells in favour of Th1 rather than Th2 cells.

The present invention utilises a specific binding molecule which bindsto Annexin-1 (Anx-A1).

Annexins are a group of calcium- and phospholipid-binding cellularproteins and are also known as lipocortins. The annexin family has 13members, including Annexin A1, Annexin A2 and Annexin A5. Annexin-A1 isalso known as Annexin-1 and is referred to herein as “Anx-A1”. Annexin-1(Anx-A1) is a 37-kDa protein and was originally described as a mediatorof the actions of glucocorticoids. Over the last few years evidence hasshown than Anx-A1 plays a homeostatic role in the adaptive immunesystem, in particular T cells, by modulating the strength of T cellreceptor (TCR) signalling. Anx-A1 acts as an endogenous down-regulatorof inflammation in cells of the innate immune system in vivo. FIG. 1A isa ribbon diagram showing the three-dimensional structure of Anx-A1.

There are eight human nucleotide sequences which encode Anx-A1. Ofthese, only four are translated and thus there are four isoforms ofAnx-A1, designated ANXA1-002, ANXA1-003, ANXA1-004 and ANXA1-006. Thesesequences are available from the Ensembl website (www.ensembl.org) andare designated OTTHUMT00000052664 (ANXA1-002), OTTHUMT00000052665(ANXA1-003), OTTHUMT00000052666 (ANXA1-004) and OTTHUMT00000052668(ANXA1-006). The amino acid and nucleotide sequences of one isoform ofhuman Annexin-1 (Anx-A1), ANXA1-003, are shown in FIG. 2 a. The aminoacid sequences of isoforms ANXA1-002, ANXA1-004 and ANXA1-006 are shownin FIGS. 2 b, 2 c and 2 d respectively. As can be seen from FIG. 2,isoforms ANXA1-002, ANXA1-004 and ANXA1-006 are either short splicevariants of ANXA1-003 or variants of ANXA1-003 with a small number ofamino acid changes.

A number of studies have shown that an N-terminal peptide of Anx-A1named Ac.2-26 acts as a bioactive surrogate of the whole protein (seee.g. Lim et al., Proc Natl Acad Sci U S A95, 14535-9, 1998).

FIG. 1B is a schematic representation of the annexin repeats and thelocation of this bioactive sequence. Peptide Ac.2-26 is an acetylatedpeptide having the sequence of amino acid residues 2-26 of thefull-length amino acid sequence of Anx-A1 shown in FIG. 2. The sequenceof peptide Ac.2-26 is shown in FIG. 1C and is as follows:

-   -   CH₃CO-AMVSEFLKQAWFIENEEQEYVQTVK

Anx-A1 and its N-terminal derived bioactive peptides mediate theirbiological effects through members of the formyl peptide receptor (FPR)family. Anx-A1 exerts its counterregulatory actions on neutrophilextravasation and innate immunity by direct binding and activation ofone member of this family, formyl peptide receptor like-1 (FPRL-1). Thepresent inventors have previously found that stimulation of T cells inthe presence of hrAnx-A1 increases T cell activation via stimulation ofFPRL-1 (D′Acquisto et al., Blood 109: 1095-1102, 2007).

The present invention utilises a specific binding molecule which bindsto Annexin-1 (Anx-A1). The Anx-A1 to which the specific binding moleculebinds is typically human Anx-A1 having the polypeptide sequence shown inFIG. 2 a, or a variant or fragment thereof, such as one of the isoformsof human Anx-A1 having the polypeptide sequence shown in FIG. 2 b, FIG.2 c or FIG. 2 d. The fragment of human Anx-A1 to which the specificbinding molecule binds is typically the polypeptide having the sequenceshown in FIG. 1C. The Anx-A1 to which the specific binding moleculebinds is typically encoded by the nucleotide sequence shown in FIG. 2 a.

As used herein the term “variant” relates to proteins which have asimilar amino acid sequence and/or which retain the same function. Forinstance, the term “variant” encompasses proteins or polypeptides whichinclude one or more amino acid additions, deletions, substitutions orthe like. Amino acid substitutions are typically conservativesubstitutions, i.e. the substitution of an amino acid with another withgenerally similar properties, such that the overall functioning islikely not to be seriously affected.

Thus the amino acids glycine, alanine, valine, leucine and isoleucinecan often be substituted for one another (amino acids having aliphaticside chains). Of these possible substitutions it is preferred thatglycine and alanine are used to substitute for one another (since theyhave relatively short side chains) and that valine, leucine andisoleucine are used to substitute for one another (since they havelarger aliphatic side chains which are hydrophobic). Other amino acidswhich can often be substituted for one another include: phenylalanine,tyrosine and tryptophan (amino acids having aromatic side chains);lysine, arginine and histidine (amino acids having basic side chains);aspartate and glutamate (amino acids having acidic side chains);asparagine and glutamine (amino acids having amide side chains); andcysteine and methionine (amino acids having sulphur containing sidechains).

Using the three letter and one letter codes the amino acids may bereferred to as follows: glycine (G or Gly), alanine (A or Ala), valine(V or Val), leucine (L or Leu), isoleucine (I or Ile), proline (P orPro), phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W orTrp), lysine (K or Lys), arginine (R or Arg), histidine (H or His),aspartic acid (D or Asp), glutamic acid (E or Glu), asparagine (N orAsn), glutamine (Q or Gln), cysteine (C or Cys), methionine (M or Met),serine (S or Ser) and Threonine (T or Thr). Where a residue may beaspartic acid or asparagine, the symbols Asx or B may be used. Where aresidue may be glutamic acid or glutamine, the symbols Glx or Z may beused. References to aspartic acid include aspartate, and references toglutamic acid include glutamate, unless the context specifies otherwise.

Amino acid deletions or insertions may also be made relative to theamino acid sequence of the protein referred to above. Thus, for example,amino acids which do not have a substantial effect on the activity ofthe polypeptide, or at least which do not eliminate such activity, maybe deleted. Such deletions can be advantageous since the overall lengthand the molecular weight of a polypeptide can be reduced whilst stillretaining activity. This can enable the amount of polypeptide requiredfor a particular purpose to be reduced—for example, dosage levels can bereduced.

Amino acid insertions relative to the sequence of the fusion proteinabove can also be made. This may be done to alter the properties of asubstance (e.g. to assist in identification, purification orexpression).

Amino acid changes relative to the sequence given above can be madeusing any suitable technique e.g. by using site-directed mutagenesis orsolid state synthesis.

It should be appreciated that amino acid substitutions or insertionswithin the scope of the present invention can be made using naturallyoccurring or non-naturally occurring amino acids. Whether or not naturalor synthetic amino acids are used, it is preferred that only L-aminoacids are present.

One can use a program such as the CLUSTAL program to compare amino acidsequences. This program compares amino acid sequences and finds theoptimal alignment by inserting spaces in either sequence as appropriate.It is possible to calculate amino acid identity or similarity (identityplus conservation of amino acid type) for an optimal alignment. Aprogram like BLASTx will align the longest stretch of similar sequencesand assign a value to the fit. It is thus possible to obtain acomparison where several regions of similarity are found, each having adifferent score. Both types of identity analysis are contemplated in thepresent invention.

Variants of the proteins and polypeptides described herein should retainthe function of the original protein or polypeptide. Alternatively or inaddition to retaining the function of the original protein orpolypeptide, variants of the proteins and polypeptides described hereintypically have at least 60% identity (as discussed above) with theproteins or polypeptides described herein, in particular the polypeptidesequences shown in FIG. 1C or FIG. 2. Typically, variants for use in theinvention have at least 70%, at least 80%, at least 90%, at least 95%,at least 97% or at least 99% identity to the proteins or polypeptidesdescribed herein, in particular the polypeptide sequences shown in FIG.1C or FIG. 2.

The percent identity of two amino acid sequences or of two nucleic acidsequences is determined by aligning the sequences for optimal comparisonpurposes (e.g., gaps can be introduced in the first sequence for bestalignment with the sequence) and comparing the amino acid residues ornucleotides at corresponding positions. The “best alignment” is analignment of two sequences which results in the highest percentidentity. The percent identity is determined by the number of identicalamino acid residues or nucleotides in the sequences being compared(i.e., % identity =number of identical positions/total number ofpositions x 100).

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm known to those of skill inthe art. An example of a mathematical algorithm for comparing twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA90:5873-5877. The NBLAST and XBLAST programsof Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporatedsuch an algorithm. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilised as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can beused to perform an iterated search which detects distant relationshipsbetween molecules (Id.). When utilising BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.Another example of a mathematical algorithm utilised for the comparisonof sequences is the algorithm of Myers and Miller, CABIOS (1989). TheALIGN program (version 2.0) which is part of the CGC sequence alignmentsoftware package has incorporated such an algorithm. Other algorithmsfor sequence analysis known in the art include ADVANCE and ADAM asdescribed in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5;and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci.85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search.

In an alternative approach, the variants can be fusion proteins,incorporating moieties which render purification easier, for example byeffectively tagging the desired protein or polypeptide. It may benecessary to remove the “tag” or it may be the case that the fusionprotein itself retains sufficient functionality to be useful.

A “specific binding molecule which binds to Anx-A1” as used herein is amolecule which binds with greater affinity to Anx-A1 than to othermolecules, i.e. which binds specifically to Anx-A1. Specific bindingmolecules which bind to Anx-A1 include anti-Anx-A1 antibodies andaptamers. The anti-Anx-A1 antibodies for use in the present inventionfunction by blocking the activation of T cells and thus, whenadministered, can be used in the treatment of T cell-mediated diseases,which are typically caused by aberrant T cell activation.

Anti-Anx-A1 antibodies can be raised, for example, against human Anx-A1having the amino acid sequence set out in FIG. 2, typically the aminoacid sequence set out in FIG. 2 a. Alternatively, anti-Anx-A1 antibodiescan be directed to a particular epitope or epitopes of human Anx-A1having the amino acid sequence set out in FIG. 2, typically the aminoacid sequence set out in FIG. 2 a. For example, anti-Anx-A1 antibodiescan be directed against an N-terminal fragment of Anx-A1, for example anN-terminal fragment of at least 188, 100, 50 or 25 amino acid residuesfrom the N-terminus of the amino acid sequence set out in FIG. 2 a.Typically, the anti-Anx-A1 antibody for use in the invention is anantibody against the N-terminal fragment of Anx-A1 termed Ac2-26 andwhich has the sequence shown in FIG. 1C, or against a fragment of atleast 6 amino acids thereof Specific binding molecules which bind toAnx-A1 therefore include anti-Anx-A1 antibodies which are antibodiesagainst the Anx-A1 fragment Ac2-26 having the sequence shown in FIG. 1Cor a fragment of at least 6 amino acids thereof In this embodiment, theanti-Anx-A1 antibody is raised against a fragment of the sequence shownin FIG. 1C which is antigenic and capable of stimulating the productionof antibodies which, when administered, can be used in the treatment ofT cell-mediated diseases, which are typically caused by aberrant T cellactivation.

As stated above, an active subfragment of the specified sequence may beused as defined. Active subfragments may consist of or include afragment of at least 6 continuous amino acid residues (a hexapeptide) ofthe N-terminal fragment of Anx-A1 termed Ac2-26 having the sequence setout in FIG. 1C, including one or more of:

AMVSEF  MVSEFL   VSEFLK    SEFLKQ     EFLKQA      FLKQAW       LKQAWF       KQAWFI         QAWFIE          AWFIEN           WFIENE           FIENEE             IENEEQ              ENEEQE              NEEQEY                EEQEYV                 EQEYVQ                 QEYVQT                   EYVQTV                   YVQTVK

Active subfragments may consist of or include a fragment of more than 6continuous amino acid residues of the N-terminal fragment of Anx-A1termed Ac2-26 having the sequence set out in FIG. 1C, for example afragment of at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at leat 20, at least 21, at least22, at least 23 or at least 24 amino acids of the sequence set out inFIG. 1C.

Anti-Anx-A1 antibodies include monoclonal and polyclonal antibodies.Typically, the anti-Anx-A1 antibody is a monoclonal antibody. Theanti-Anx-A1 antibody can be a commercially available antibody, forexample a rabbit polyclonal or mouse monoclonal antibody. Typically, theanti-Anx-A1 antibody is humanised, as described in detail below.

Monoclonal antibodies can be produced from hybridomas. These aretypically formed by fusing myeloma cells and spleen cells which producethe desired antibody in order to form an immortal cell line. Thewell-known Kohler & Milstein technique (Nature 256:495-497 (1975)) orsubsequent variations upon this technique can be used to produce amonoclonal antibody for use in accordance with the invention.

Polyclonal antibodies can be raised by stimulating their production in asuitable animal host (e.g. a mouse, rat, guinea pig, rabbit, sheep, goator monkey) by injection of Anx-A1, or a variant or fragment thereof,into the animal. If desired, an adjuvant may be administered togetherwith the Anx-A1 protein. Well-known adjuvants include Freund's adjuvant(complete and incomplete) and aluminium hydroxide. The antibodies canthen be purified by virtue of their binding to Anx-A1.

Techniques for producing monoclonal and polyclonal antibodies that bindto a particular polypeptide/protein are now well developed in the artand are discussed in standard immunology textbooks, for example in Roittet al, Immunology second edition (1989), Churchill Livingstone, London.

In addition to whole antibodies, the present invention includesderivatives thereof which are capable of binding to Anx-A1 as describedherein. Thus the present invention includes antibody fragments andsynthetic constructs. Examples of antibody fragments and syntheticconstructs are given by Dougall et al in Trends Biotechnol., 12: 372-379(1994).

Antibody fragments include, for example, Fab, F(ab')₂ and Fv fragments.Fab fragments are discussed in Roitt et al [supra]. Fv fragments can bemodified to produce a synthetic construct known as a single chain Fv(scFv) molecule. This includes a peptide linker covalently joiningvariable heavy chain (V_(H)) and variable light chain (V_(L)) regions,which contributes to the stability of the molecule. The linker maycomprise from 1 to 20 amino acids, such as for example 1, 2, 3 or 4amino acids, 5, 10 or 15 amino acids, or other intermediate numbers inthe range 1 to 20 as convenient. The peptide linker may be formed fromany generally convenient amino acid residues, such as glycine and/orserine. One example of a suitable linker is Gly₄Ser. Multimers of suchlinkers may be used, such as for example a dimer, a trimer, a tetrameror a pentamer, e.g. (Gly₄Ser)₂, (Gly₄Ser)₃, (Gly₄Ser)₄ or (Gly₄Ser)₅.However, in other embodiments no peptide linker is present and the V_(L)domain is linked to the V_(H) domain by a peptide bond.

The specific binding molecule may be an analogue of a single-chainvariable fragment (scFv). For example, the scFv may be linked to otherspecific binding molecules (for example other scFvs, Fab antibodyfragments and chimeric IgG antibodies (e.g. with human frameworks)). ThescFv may be linked to other scFvs so as to form a multimer which is amulti-specific binding protein, for example a dimer, a trimer or atetramer. Bi-specific scFv's are sometimes referred to as diabodies,tri-specific as triabodies and tetra-specific as tetrabodies.

An scFv can be prepared by any suitable technique using standardchemical or molecular biology techniques. In one embodiment of theinvention, the monoclonal antibody analogues can be prepared as scFv'sfrom a nave human antibody phage display library (McCafferty et al.,Nature 348, 552-554 (1990); and as described in WO 92/01047).

Other synthetic constructs that can be used include ComplementarityDetermining Region (CDR) peptides. These are synthetic peptidescomprising antigen-binding determinants Peptide mimetics can also beused. These molecules are usually conformationally restricted organicrings that mimic the structure of a CDR loop and that includeantigen-interactive side chains.

Synthetic constructs include chimeric molecules. Thus, humanisedantibodies or derivatives thereof are within the scope of the antibodiesfor use in the present invention. Methods for humanising antibodies arewell known in the art. The antibody can be humanised by modifying theamino acid sequence of the antibody. An example of a humanised antibodyis an antibody having human framework regions, but rodent (for examplemurine) hypervariable regions. Ways of producing chimeric antibodies arediscussed for example by Morrison et al in PNAS, 81: 6851-6855 (1984)and by Takeda et al in Nature, 314: 452-454 (1985). Humanisation can beperformed, for example, as described by Jones et al in Nature, 321:522-525 (1986); Verhoeyen et al in Science, 239: 1534-1536; Riechmann etal in Nature 332: 323-327, 1988. Methods to reduce the immunogenicity ofthe specific binding molecules of the invention may include CDR graftingon to a suitable antibody framework scaffold or variable surface residueremodelling, e.g. by site-directed mutagenesis or other commonly usedmolecular biological techniques (Roguska et al Protein Eng. 9 895-904(1996)).

Other methods applicable include the identification of potential T-cellepitopes within the molecule, and the subsequent removal of these e.g.by site-directed mutagenesis (de-immunisation). Humanisation of the CDRregions or of the surrounding framework sequence may be carried out asdesired.

Synthetic constructs also include molecules comprising an additionalmoiety that provides the molecule with some desirable property inaddition to antigen binding. For example the moiety may be a label (e.g.a fluorescent or radioactive label). Alternatively, it may be apharmaceutically active agent.

The present invention relates to the use of a specific binding moleculewhich binds to Anx-A1 for the treatment of T cell-mediated disease.

The present invention can be used to treat a wide range of diseaseswhich are mediated by T cells. In the present context, “T cell-mediateddisease” means any disease or condition in which T cells play a role inpathogenesis or development of the disease or condition. T cell-mediateddiseases are typically caused by aberrant T cell activation.Accordingly, such diseases can be treated by preventing the activationof T cells by blocking the activity of Anx-A1. Typically, the Tcell-mediated diseases treated in the present invention are diseases inwhich Th1 cells play a role.

T cell-mediated diseases include but are not limited tograft-versus-host disease, graft rejection, atherosclerosis, HIV and/orAIDS, psoriasis and some autoimmune diseases. Autoimmune diseases whichcan be treated according to the present invention include rheumatoidarthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus(SLE), Addison's disease, Grave's disease, scleroderma, polymyositis,some forms of diabetes mellitus (for example juvenile onset diabetes),autoimmune uveoretinitis, ulcerative colitis, pemphigus vulgaris,inflammatory bowel disease and autoimmune thyroiditis. The Tcell-mediated disease is typically rheumatoid arthritis, multiplesclerosis, systemic lupus erythematosus or atherosclerosis.

The T cell-mediated disease is typically rheumatoid arthritis. Inrheumatoid arthritis (RA), it is thought that T cells recognise andinteract with antigen presenting cells in the synovium. Once activated,these cells produce cytokines and effector molecules; this sequential,expanded production of cytokines constitutes the “cytokine cascade” thatresults in the activation of macrophages and induction of theinflammatory process, culminating in degradation and resorption ofcartilage and bone. Over time, bone erosion, destruction of cartilage,and complete loss of joint integrity can occur. Eventually, multipleorgan systems may be affected.

In another embodiment, the T cell-mediated disease is atherosclerosis.Inflammation plays a key role in coronary artery disease and othermanifestations of atherosclerosis. Immune cells dominate earlyatherosclerotic lesions, their effector molecules accelerate progressionof the lesions, and activation of inflammation can elicit acute coronarysyndromes. Adaptive immunity is highly involved in atherogenesis sinceit has been shown to interact with metabolic risk factors to initiate,propagate, and activate lesions in the arterial tree.

Two mouse models with features of hypercholesterolemia and rapiddevelopment of atherosclerosis, the ApoE^(−/−) and the low-densitylipoprotein receptor-knockout mouse (LDLR^(−/−)), are useful in thestudy of atherosclerosis as they mimic the cellular composition of humanlesions, particularly in content of T lymphocytes. Lymphocyterecruitment is increased in the arteries of the atherosclerotic-proneApoE^(−/−) mice even well before the onset of the pathology.

The presence of T-lymphocytes has functional consequences as theircomplete absence reduces lesion formation during moderatehypercholesterolemia. CD4+ IFN-γ-secreting type-1 helper (Th1) cells arethe predominant type of T cell found in plaques, and these T cells exertpro-atherogenic and plaque-destabilising effects.

The inventors have now found that Anx-A1 is expressed in both human andmurine atherosclerotic plaques and that there is a correlation betweenAnx-A1 expression and MS in a mouse model.

In another embodiment, the T cell-mediated disease is systemic lupuserythematosus (SLE). The inventors have now found that Anx-A1 mRNA andprotein are expressed at higher levels in T cells from SLE patients thanin T cells from healthy volunteers.

In relation to the ability of Anx-A1 to favour differentiation of Th1cells, the present invention can also be used, for example, to limituncontrolled protective cellular (Th1) responses against intracellularpathogens and to treat extracellular infection (Th2 response) bysuppressing Th1 differentiation and favouring Th2 differentiation.

The specific binding molecule which binds to Anx-A1 is typicallyformulated for use with a pharmaceutically acceptable carrier,excipient, vehicle, adjuvant and/or diluent. The present invention thusencompasses a pharmaceutical composition comprising a specific bindingmolecule which binds to Annexin-1 (Anx-A1) for use in the treatment of Tcell-mediated disease. The pharmaceutical composition comprises aspecific binding molecule which binds to Annexin-1 (Anx-A1) and apharmaceutically acceptable carrier, excipient, vehicle, adjuvant and/ordiluent. Such compositions may be prepared by any method known in theart of pharmacy, for example by admixing the active ingredient with thecarrier, excipient, vehicle, adjuvant and/or diluent under sterileconditions.

Suitable carriers, vehicles, adjuvants and/or diluents are well known inthe art and include saline, phosphate buffered saline (PBS),carboxymethylcellulose (CMC), methylcellulose,hydroxypropylmethylcellulose (HPMC), dextrose, liposomes, polyvinylalcohol, pharmaceutical grade starch, mannitol, lactose, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose (andother sugars), magnesium carbonate, gelatin, oil, alcohol, detergents,emulsifiers or water (preferably sterile). The specific binding moleculewhich binds to Anx-A1 can be formulated as a liquid formulation, whichwill generally consist of a suspension or solution of the specificbinding molecule which binds to Anx-A1 in a suitable aqueous ornon-aqueous liquid carrier or carriers, for example water, ethanol,glycerine, polyethylene glycol (PEG) or an oil.

Typically, when the specific binding molecule which binds to Anx-A1 isan antibody, the antibody is PEGylated, i.e. covalently attached to apolyethylene glycol. Typically, this has the effect of reducing theimmunogenicity and increasing the half-life of said antibody.

The specific binding molecule which binds to Anx-A1 can be administeredalone or together with another agent.

The specific binding molecule which binds to Anx-A1 for use in thepresent invention is typically administered to a subject in atherapeutically effective amount. Such an amount is an amount effectiveto ameliorate, eliminate or prevent one or more symptoms of Tcell-mediated disease. Preferably, the subject to be treated is a human.However, the present invention is equally applicable to human orveterinary medicine. For example, the present invention may find use intreating companion animals, such as dogs and cats, or working animals,such as race horses.

The specific binding molecule which binds to Anx-A1 can be administeredto the subject by any suitable means. The specific binding moleculewhich binds to Anx-A1 can be administered systemically, in particularintra-articularly, intra-arterially, intraperitoneally (i.p.),intravenously or intramuscularly. However, the specific binding moleculewhich binds to Anx-A1 can also be administered by other enteral orparenteral routes such as by subcutaneous, intradermal, topical(including buccal, sublingual or transdermal), oral (including buccal orsublingual), nasal, vaginal, anal, pulmonary or other appropriateadministration routes.

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; as powders orgranules; as solutions, syrups or suspensions (in aqueous or non-aqueousliquids; or as edible foams or whips; or as emulsions). Suitableexcipients for tablets or hard gelatine capsules include lactose, maizestarch or derivatives thereof, stearic acid or salts thereof Suitableexcipients for use with soft gelatine capsules include for examplevegetable oils, waxes, fats, semi-solid, or liquid polyols etc. For thepreparation of solutions and syrups, excipients which may be usedinclude for example water, polyols and sugars. For the preparation ofsuspensions, oils (e.g. vegetable oils) may be used to provideoil-in-water or water in oil suspensions.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active ingredient may be delivered from the patch byiontophoresis as generally described in Pharmaceutical Research,3(6):318 (1986).

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils. For infections of theeye or other external tissues, for example mouth and skin, thecompositions are preferably applied as a topical ointment or cream. Whenformulated in an ointment, the active ingredient may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredient may be formulated in a cream with an oil-in-watercream base or a water-in-oil base. Pharmaceutical compositions adaptedfor topical administration to the eye include eye drops wherein theactive ingredient is dissolved or suspended in a suitable carrier,especially an aqueous solvent. Pharmaceutical compositions adapted fortopical administration in the mouth include lozenges, pastilles andmouth washes.

Pharmaceutical compositions adapted for rectal administration may bepresented as suppositories or enemas.

Pharmaceutical compositions adapted for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size forexample in the range 20 to 500 microns which is administered in themanner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable compositions wherein the carrier is a liquid, foradministration as a nasal spray or as nasal drops, include aqueous oroil solutions of the active ingredient.

Pharmaceutical compositions adapted for administration by inhalationinclude fine particle dusts or mists which may be generated by means ofvarious types of metered dose pressurised aerosols, nebulizers orinsufflators.

Pharmaceutical compositions adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solution which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation substantially isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Excipients which may beused for injectable solutions include water, alcohols, polyols,glycerine and vegetable oils, for example. The compositions may bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carried, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules and tablets.

The pharmaceutical compositions may contain preserving agents,solubilising agents, stabilising agents, wetting agents, emulsifiers,sweeteners, colourants, odourants, salts, buffers, coating agents orantioxidants. They may also contain therapeutically active agents inaddition to the specific binding molecule which binds to Anx-A1.

The dose of the specific binding molecule which binds to Anx-A1 to beadministered may be determined according to various parameters,especially according to the specific binding molecule which binds toAnx-A1 used; the age, weight and condition of the patient to be treated;the route of administration; and the required regimen. A physician willbe able to determine the required route of administration and dosage fora particular patient.

This dosage may be repeated as often as appropriate. If side effectsdevelop the amount and/or frequency of the dosage can be reduced, inaccordance with normal clinical practice.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage of the active agent will be from 1 μg/kg to10mg/kg body weight, typically around 10 μg/kg to 1 mg/kg body weight.The physician in any event will determine the actual dosage which willbe most suitable for an individual which will be dependant on factorsincluding the age, weight, sex and response of the individual. The abovedosages are exemplary of the average case. There can, of course, beinstances where higher or lower dosages are merited, and such are withinthe scope of this invention

In a second aspect of the invention, there is provided the use of aspecific binding molecule which binds to Anx-A1 in the manufacture of amedicament for the treatment of T cell-mediated disease.

In a third aspect of the invention, there is provided a method for thetreatment of T cell-mediated disease comprising administering to asubject in need thereof a therapeutic amount of a specific bindingmolecule which binds to Anx-A1. As stated above, the method of treatmentmay of a human or an animal subject and the invention extends equally tomethods of treatment for use in human and/or veterinary medicine.

Preferred features for the second and third aspects of the invention areas for the first aspect mutatis mutandis.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described by way of reference to thefollowing Examples and Figures which are provided for the purposes ofillustration only and are not to be construed as limiting on theinvention. Reference is made to a number of Figures, in which:

FIG. 1A is a ribbon diagram of annexin-1 structure showing the fourannexin repeats and the N-terminal domain. FIG. 1B is a schematicrepresentation of the annexin repeats and the location of the bioactivesequence, Annexin-1 peptide Ac.2-26. FIG. 1C shows the amino acidsequence of peptide Ac.2-26, which is an acetylated N-terminal peptidefragment of Anx-A1.

FIG. 2 a shows (i) the amino acid sequence and (ii) the nucleotidesequence of human Annexin-1 (Anx-A1), isoform ANXA1-003. FIG. 2 b showsthe amino acid sequence of human Annexin-1 (Anx-A1), isoform ANXA1-002.

FIG. 2 c shows the amino acid sequence of human Annexin-1 (Anx-A1),isoform ANXA1-004. FIG. 2 d shows the amino acid sequence of humanAnnexin-1 (Anx-A1), isoform ANXA1-006.

FIGS. 3A-3D show the effect of human recombinant Annexin-1 (hrAnx-A1) onT cell activation. Pre-treatment of murine nave CD4+ primary cells withhrAnx-A1 followed by activation with different concentrations ofanti-CD3/CD28 augmented cell proliferation (FIG. 3A), IL-2 production(FIG. 3B) and cell surface expression of CD25 and CD69 (FIGS. 3C and3D).

FIGS. 4A-4D show that endogenous Anx-A1 modulates T cell proliferation.Stimulation of Anx-A1^(+/+) or Anx-A1^(−/−) T cells with anti-CD3,anti-CD3/CD28 or PMA/Ionomycin showed a decrease rate of ³H-thymidineincorporation (FIGS. 4A, 4B and 4C, respectively) and IL-2 production(FIG. 4D) in the Anx-A1 deficient T cells compared to controlunstimulated T cells.

FIGS. 5A and 5B show activation of Activator Protein-1 (AP-1), NuclearFactor-κB (NF-κB) and Nuclear Factor of Activated T cells (NFAT) in thepresence or absence of Anx-A1 (FIG. 5A), and a comparison of theactivation of AP-1, NF-κB and NFAT in Anx-A1^(−/+) and Anx^(−/−)T cells(FIG. 5B).

FIG. 6A shows FACS analysis of FPRL-1 expression in T cells stimulatedwith anti-CD3/CD28 (5.0 μg/ml) for the indicated times. FIG. 6B showsthe cellular localization of Anx-A1 in T cells before (Control) or afterstimulation with anti-CD3/CD28 (5.0 μg/ml). FIG. 6C is a schematicrepresentation of the role of the Anx-A1/FPRL-1 system in T cells.

FIGS. 7A and 7B show that exogenous and endogenous Anx-A1 modulatesTh1/Th2 differentiation. FIG. 7A shows the results when nave lymph nodeT cells were differentiated in vitro in Th1 (black bars) or Th2 (whitebars) conditions in presence or absence of hrAnx-A1 and thenrestimulated with platebound anti-CD3 to measure Th1 or Th2 cytokineproduction. FIG. 7B shows the results when nave lymph node T cells fromAnx-A1^(+/+) or Anx-A1^(−/−) mice were differentiated in vitro in Th1(first and second column graphs from the left) or Th2 (third and fourthcolumn graphs from the left) conditions and then restimulated withplatebound anti-CD3 to measure Th1 or Th2 cytokine production.

FIG. 8A and 8B paw volume (FIG. 8A) and clinical score (FIG. 8B) of DBAmice treated with PBS or hrAnx-A1 for 12 days during the immunizationphase of the collagen-induced arthritis (CIA) model. FIG. 8C is ananalysis of Anx-A1 expression in CD4+ cells of healthy controlvolunteers (HC) or rheumatoid arthritis (RA) patients. FIG. 8D shows animmunohistochemical analysis of Anx-A1 expression in synovial tissuefrom RA patients.

FIG. 9 shows the effects of full length hrAnx-A1 and the N-terminalpeptide Ac 2-26 on T cell activation.

FIGS. 10A and 10B show the expression of Anx-A1 in human atheroscleroticplaques Immunohistochemical analysis of Anx-A1 expression with mousemonoclonal anti-human Anx-A1 antibody 1B (FIG. 10A) or with nonimmuneIgG (FIG. 10B) in carotid atherosclerotic plaques removed from patientsduring carotid endarteretomy surgery. Photographs are from a singlepatient and representative of six different patients with similarconditions.

FIGS. 11A-11C show expression of Anx-A1 in murine atheroscleroticplaques. FIG. 11 shows immunofluorescence visualization of Anx-A1 inApoE^(−/−) mice aortic sinus (FIG. 11A and FIG. 11B) and brachiocefalicartery (FIG. 11C). Sections were stained with Dapi to locate nuclei.Results illustrated are from a single experiment and are representativeof three separate experiments. Original magnification: ×200 (FIG. 11Aand FIG. 11B), ×400 (FIG. 11C).

FIG. 12 shows expression of Annexin-1 in Systemic Lupus Erythematosus(SLE) patients. RT-PCR (upper panel) and Western Blot (lower panel)analysis of Anx-A1 expression in T cells from healthy (Control) orSystemic Lupus Erythematosus (SLE) patients. The numbers in the figureindicate the volume (p1) of cDNA or the amount (pg) of proteins obtainedfrom the same number (2×10⁶) of T cells collected from healthy (Control)or Systemic Lupus Erythematosus (SLE) patients.

FIG. 13 shows inhibition of activation of the T cell receptor (TCR),measured in terms of interleukin-2 (IL-2) production, in humanperipheral T cells from one donor incubated with a neutralisingmonoclonal antibody raised against human recombinant annexin-1(anti-AnxA1 mAblA).

FIG. 14 shows inhibition of activation of the T cell receptor (TCR),measured in terms of interleukin-2 (IL-2) production, in humanperipheral T cells from a different donor incubated with a neutralisingmonoclonal antibody raised against human recombinant annexin-1(anti-AnxA1 mAblA).

FIGS. 15A and 15B show spinal cord sections from C57BL/6 mice immunizedwith MOG₃₅₋₅₅ and CFA and from which spinal cords removed at day 12(score 0), day 18 (score 2) and day 20 (score 4). The sections werestained with hematoxylin and eosin (H&E, FIG. 15A) or anti-AnxA1 (FIG.15B). For each staining, the right panels (20×) show a highermagnification of an area of the left panels (4X). Results representativeof 3 experiments.

FIG. 16 shows spinal cord sections from C57BL/6 mice immunized withMOG₃₅₋₅₅ and CFA and from which spinal cords removed at day 20 (score4). The sections were stained with anti-AnxA1 and anti-CD3 (A) oranti-F4/80 (B). The right panels show an overlay of the two singlestainings on the right. Results representative of 3 experiments.

FIG. 17 shows the results from a study in which C57BL/6 mice wereimmunized with MOG₃₅₋₅₅ and CFA and monitored daily for signs andsymptoms of EAE (A) or weight gain/loss (B) for 23 days. Results aremeans±SEM (n=10/group). ** p<0.01, representative of 3 experiments.

FIG. 18 shows the incorporation of ³H-Thymidine (A) and the productionof IL-2 (B) of lymph node cells obtained from AnxA1^(+/+) andAnxA1^(−/−) mice immunized with MOG₃₅₋₅₅ and CFA and sacrificed after 14days. Cells were stimulated with MOG₃₅₋₅₅ for 48 hours and pulsed with 1μCi ³H-Thymidine for 12 hours. Cell supernatants were used to measureIL-2 production. Results are means±SEM (n=4/group). * p<0.05, ** p<0.01,representative of 3 experiments.

FIG. 19 shows the total cell number of spleen (A) and lymph node (B)cells obtained from AnxA1^(+/+) and AnxA1^(−/−) mice immunized withMOG₃₅₋₅₅ and CFA and sacrificed after 14 days. C and D show thecytofluorimetric analysis of lymph node cells with anti-CD4 FITC andanti-CD8 PE. Results are means±SEM (n=10/group). ** p<0.01,representative of 3 experiments.

FIG. 20 shows levels of (A) IFN-γ, (B) IL-2, (C) TNF-α and (D) IL-17 inthe cell supernatants of lymph node cells obtained from AnxA1^(+/+) andAnxA1^(−/−) mice immunized with MOG₃₅₋₅₅ and CFA and sacrificed after 14days. Cells were stimulated with the indicated concentration of MOG₃₅₋₅₅for 4 days and the supernatants used for cytokine ELISA. Results aremeans±SEM (n=4/group). * p<0.05, ** p<0.01, representative of 3experiments.

FIG. 21 shows haematoxylin-eosin staining of spinal cord sectionsobtained from AnxA1^(+/+) (A) and AnxA1^(−/−) (B) mice immunized withMOG₃₅₋₅₅ and CFA and sacrificed after 22 days. For each staining, theright panels (20X) show a higher magnification of an area of the leftpanels (4X). Consecutive sections were stained with anti-CD3 (C) oranti-F4/80 (D). Pictures are representative of three separateexperiments with similar results.

FIG. 22 shows FACS analysis of CD3 (A) and F4/80 (B) positivemononuclear cells recovered by Percoll gradient of spinal cordhomogenates obtained from AnxA1^(+/+) and AnxA1^(−/−) mice immunizedwith MOG₃₅₋₅₅ and CFA and sacrificed after 14 days. The dot plots andhistograms are from a single mouse and representative of 2 experimentswith n=4 mice. The numbers in the histograms indicate the percentage ofCD3⁺ and F4/80⁺ cells.

EXAMPLES 1 TO 10

Materials and Methods

Reagents

Anti-mouse CD3 (clone 145-2C11), anti-mouse CD28 (clone 37.51),anti-human CD3 (clone OKT3), anti-human CD28 (clone CD28.2),PE-conjugated anti-CD69 (clone H1.2F3), FITC-conjugated anti-CD25 (clonePC61.5), murine IL-2, IL-4, IFN-γ, IL-12, antiIL-4 (clone 11B11), andantiIFN-₇ (clone XMG1.2) were purchased from eBioscience (Wembley,United Kingdom). Endotoxin-free human recombinant Anx-A1 (hrAnx-A1) wasprepared as described. In some experiments, we used denatured hrAnx-A1(heat-inactivated at 95° C. for 5 minutes) as positive control. Unlessotherwise specified, all the other reagents were from Sigma-Aldrich (StLouis, Mo.).

Mice

BALB/C, C57/BL6 and DBA/1 male mice were obtained from Charles RiverLaboratories (Wilmington, Mass.). Annexin 1 null mice on BALB/C weregenerated in our lab and bred in pathogen-free conditions in our animalfacilities. All mice used in these studies were between 6 and 8 weeksold. Animal work was performed according to United Kingdom Home Officeregulations (Guidance on the Operation of Animals, Scientific ProceduresAct 1986) and along the directives of the European Union.

Isolation of cells from Patients

Peripheral blood mononuclear cells (PBMCs) were prepared from peripheralblood using Ficoll density centrifugation (Ficoll-Paque Plus; AmershamBiosciences, Freiburg, Germany). CD4⁺ cells were selected fromperipheral blood using positive selection. Briefly, peripheral blood wassubjected to Ficoll density centrifugation (Ficoll-Paque Plus; AmershamBiosciences). Adherent cells were removed from the mononuclear cells byadherence to serum-coated plastic. Nonadherent cells were incubated withmouse anti-human CD4 antibody (RFT4), washed in buffer(phosphate-buffered saline [PBS], 0.5% bovine serum albumin [BSA], 2 mMEDTA pH 7.2) and incubated with goat antimouse antibody conjugated to amagnetic bead (Miltenyi Biotec, Auburn, Calif.). Cells were run througha MACS column (Miltenyi Biotec) and CD4⁺ cells were collected. Thepurity of the cells was assessed by flow cytometry. The medianpercentage of CD3⁺ CD4⁺ cells following the 10 depletions was 98%(range, 97%-99.3%). Remaining cells were resuspended in lysis buffer(Ambion, Huntingdon, United Kingdom).

Cell Culture

Primary murine T cells were prepared from lymph nodes by negativeselection. Briefly, axillary, inguinal, and mesenteric lymph nodes wereteased apart to make a single cell suspension, then washed and layeredover Ficoll. The buffy coat was washed 2 times and then incubated withthe antibody mix and the magnetic beads following the manufacturer'sinstructions (mouse T-cell negative isolation kit; Dynal, Bromborough,United Kingdom). In some experiments, cells were further purified toobtain naive CD62L⁺ CD4⁺ T cells by using the Miltenyi Biotec CD62L⁺CD4⁺ T-cell isolation kit. Th0 conditions were created by T cells for 4days in 6-well plates precoated with anti-CD3 (5 ng/mL) and anti-CD28 (5ng/mL) in complete RPMI medium (10% fetal calf serum [FCS], 2 mML-glutamine, and 100 units mL⁻¹ gentamycin) containing murine IL-2 (20U/mL). Th1 conditions were created with murine IL-12 (3.4 ng/mL;eBioscience), IL-2 (20 U/mL; eBioscience), and anti-IL4 (clone 11B11; 2ng/mL). Th2 conditions were created with IL-4 (3000 U/mL; Peprotech,Rocky Hill, N.J.), IL-2 (20 U/mL), and antiIFN-₇ (clone XMG1.2; 2ng/mL). Jurkat cells were obtained from ATCC (Manassas, Va.) and werecultured in complete RPMI medium.

Flow Cytometric Analysis

Purified lymph node T cells were pretreated with human recombinantAnx-A1 for 2 hours at 37° C. in Eppendorf tubes and then stimulated withplate-bound anti-CD3 and anti-CD28 as indicated in the figures. After 16hours, the cells were stained with PE-conjugated anti-CD69 (cloneH1.2F3) and FITC-conjugated anti-CD25 (clone PC61.5) diluted in FACSbuffer (PBS containing 1% FCS and 0.02% NaN₂). Intact cells were gatedby using forward and side scatter and were analyzed with the CellQuestprogram (Becton Dickinson, Franklin Lakes, N.J.) on a FACScan flowcytometer. To analyze FPRL-1 expression, human peripheral blood T cellswere stimulated with plate-bound anti-CD3 and anti-CD28 for differenttimes and thereafter stained with mouse anti-human FPRL-1 (clone6C7-3-A; 5 ug/mL), followed by FITC-conjugated antibody.

Cell Proliferation Assay

Purified lymph node T cells (105 cells/mL) were incubated with mediumalone or with different concentrations of hrAnx-A1 for 2 hours at 37° C.in Eppendorf tubes. Thereafter, aliquots of 200-0_, cell suspension werestimulated by plate-bound anti-CD3 and anti-CD28 for 24 hours in 96-wellplates. After 18 hours, cultures were pulsed for 8 hours with 1 uCi (3.7x 10⁴ Bg) [³H]-thymidine (Amersham Pharmacia Biotech, Piscataway, N.J.)and incorporated radioactivity was measured by automated scintillationcounter (Packard Instruments, Pangbourne, United Kingdom).

Electromobility Shift Assays

Nuclear extracts were harvested from 3×10⁶ to 5×10⁶ cells according topreviously described protocols. Nuclear extracts (3 μg to 5 μg) wereincubated with 1 μg (for NFAT) or 2 μg (for NF-M3 and AP-1) of poly(dI:dC) in 20 μL, binding buffer with ³²P end-labeled, double-strandedoligonucleotide probes (5×10⁵ cpm), and fractionated on a 6%polyacrylamide gel (29:1 cross-linking ratio) in 0.5% TBE for 2.5 hoursat 150 V. The NF-κB and AP-1 binding buffer (10×) contained 100 mMTris-HCl, (pH 7.5), 500 mM NaCl, 10 mM EDTA, 50% glycerol, 10 mg/mLalbumin, 30 mM GTP, 10 mM DTT. The NFAT binding buffer (10×) contained100 mM Hepes (pH 7.9), 500 mM KCl, 1 mM EDTA, 1 mM EGTA, 50% glycerol, 5mg/mL albumin, 1% Nonidet P-40, 10 mM DTT. The NF-M3 and AP-1double-stranded oligonucleotide probes were from Promega and the NFATwas from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Western Blotting Analysis

Lymph node T cells were incubated as indicated in the figures. Afterincubation at 37° C. for various time periods, cells were lysed inice-cold lysis buffer (1% NP-40, 20 mM Tris pH 7.5, 150 mM NaCl, 1 mMMgCl₂, 1 mM EGTA, 0.5 mM PMSF, 1 μM aprotinin, 1 μM leupeptin, 1 μMpepstatin, 50 mM NaF, 10 mM Na₄P₂O₇, and 1 mM NaVO₄, 1 mMB-glycerophosphate). The cell lysates were centrifuged at 13/226g (13000) rpm for 5 minutes at 4° C. and the supernatants were collected andsubjected to electrophoresis on SDS-10% polyacrylamide gel. Aftertransfer, the membranes were incubated overnight with antibodies dilutedin Tris-buffered saline solution containing Tween-20 (TTBS: 0.13 M NaCl;2.68 mM KCl; 0.019 M Tris-HCl; 0.001% vol/vol Tween-20; pH 7.4) with 5%nonfat dry milk at 4° C. For the experiments with anti-pERK1/2 andanti-Akt, the TTBS buffer was supplemented with 50 mM NaF and bovineserum albumin (5%) was used instead of milk. For each condition, extractequivalents obtained from the same number of cells were usedImmunoblotting and visualization of proteins by enhancedchemiluminescence (ECL; Amersham Pharmacia Biotech) were performedaccording to the manufacturer's instructions. To obtain cytosolic andmembrane fractions, cells were first collected and washed in ice-coldPBS and then centrifuged briefly for 2 minutes at 300 g. The resultantcell pellet was lysed in lysis buffer (20 mM Tris-HCl, pH 7.5; proteaseinhibitors as listed in the lysis buffer) and passed through a 25-gaugeneedle at least 5 times to ensure sufficient lysis. The suspension wasthen centrifuged for 2 minutes at 300 g, the supernatant collected, andcentrifuged again for 45 minutes at 800g (4° C.). At this stage thesupernatant (cytosolic fraction) was collected and the pellet (membranefraction) resuspended in lysis buffer containing 1% (vol/vol) TritonX-100. All fractions were kept on ice throughout the experiments.

Cytokine ELISA

For Th1/Th2 cytokine production analysis, Th0/Th1/Th2 cells (10⁶/mL)obtained after 4-day differentiation in skewing conditions and 1 day ofresting in complete RPMI medium, were stimulated with plate-boundanti-CD3 (5 μg/mL) for 8 h in 24-well plates. Culture supernatants werecollected and analyzed for IFN-γ, IL-2, IL-4 and IL-10 content by usingTh1/Th2 panel ELISA kit (eBioscience). The IL-13 ELISA kit was alsopurchased from eBioscience.

Example 1 Effect of Human Recombinant Annexin-1 (hrAnx-A1) on T CellActivation

Murine naive lymph nodes T cells were stimulated with 5.0 (v), 2.5 (G)and 1.25 (T) μg/ml of anti-CD3/CD28 in the absence of or in the presenceof different concentrations of hrAnx-A1 for 24 hrs and were then pulsedwith ³H-thymidine to measure proliferation. The results are shown inFIG. 3A.

FIG. 3B shows IL-2 production from primary murine nave lymph node Tcells stimulated with anti-CD3/CD28 (1.25 μg/ml) in the absence of or inthe presence of different concentrations of hrAnx-A1 for 24 hrs.

Murine nave lymph node T cells were stimulated with anti-CD3/CD28 at aconcentration of 1.25 μg/ml (left column), 2.5 μg/ml (middle column) and5.0 μg/ml (right column), in the absence of (upper panels) or in thepresence of (lower panels) hrAnx-A1 (600nM) for 12 hrs and then analyzedfor CD25 and CD69 expression by FACS. The results are shown in FIG. 3C.

In FIG. 3D, murine nave lymph node T cells were stimulated with theindicated concentration of anti-CD3/CD28 in the presence of 150 (×), 300(Ω) and 600 (μ) nM of hrAnx-A1 for 12 hours and then analysed for CD25(left graph) and CD69 (right graph) expression by FACS.

In all of the experiments values are mean±S.E. of n=3-4 mice. *P<0.05;**P<0.01.

The results show that pre-treatment of murine naive CD4+ primary cellswith hrAnx-A1 followed by activation with different concentrations ofanti-CD3/CD28 augmented cell proliferation (FIG. 3A), IL-2 production(FIG. 3B) and cell surface expression of CD25 and CD69 (FIGS. 3C and D).

Example 2 Endogenous Anx-A1 Modulates T Cell Proliferation

FIG. 4 shows that: (A) anti-CD3 (5.0 mg/ml) (B) anti-CD3/CD28 (5.0mg/ml) or (C) PMA (20 ng/ml) and Ionomycin (2 ng/ml) inducedproliferation of wild type and Anx-A1 deficient T cells, expressed as apercentage of ³H-thymidine incorporation compared to controlunstimulated T cells. In some experiments, cells were also activated inpresence of mouse recombinant IL-2 (20 ng/ml). Values are mean±S.E. ofn=4-5 t t P<0.01 vs IL-2 stimulated Anx-A1^(+/+) cells; **P<0.01 vsanti-CD3 or anti-CD3/CD28 or PMA/Ionomycin stimulated Anx-A1^(+/+)cells; §§P<0.01 vs anti-CD3 or anti-CD3/CD28 or PMA/Ionomycin stimulatedAnx-A1^(−/−) cells.

FIG. 4D shows IL-2 production from nave lymph node T cells stimulatedwith anti-CD3, anti-CD3/CD28 (5.0 mg/ml) or PMA (20 ng/ml) and Ionomycin(2 ng/ml) for 24 h. Values are mean±S.E. of n=4-5 mice. **P<0.01.

The results show that stimulation of Anx-A1^(+/+) or Anx-A1^(−/−)T cellswith anti-CD3, anti-CD3/CD28 or PMA/ionomycin showed a decrease rate of³H-thymidine incorporation (FIGS. 4A, 4B and 4C, respectively) and IL-2production (FIG. 4D) in the Anx-A1 deficient T cells compared to controlunstimulated T cells.

Example 3 Activation of AP-1, NF-κB and NFAT in Presence or Absence ofAnx-A1

Investigations were carried out into how exogenous and endogenous Anx-A1modulates T cell activation. The three major transcriptional activatorsof T cells, namely Activator Protein-1 (AP-1), Nuclear Factor-κB (NF-κB)and Nuclear Factor of Activated T cells (NFAT) were analysed in cellsstimulated in presence of hrAnx-A1.

FIG. 5A is an Electrophoretic Mobility Shift Assay for AP-1, NF-κB andNFAT activation in T cells stimulated with anti-CD3/CD28 (1.25 μg/ml) inpresence or absence of the indicated concentration of hrAnx-A1 . FIG. 5Bshows a comparison of the activation of AP-1, NF-κB and NFAT inAnx-A1^(+/+) and Anx^(−/−)T cells stimulated with anti-CD3/CD28 (5.0μg/ml).

The results demonstrated increased activation of all three transcriptionfactors (FIG. 5A). Conversely, Anx-A1^(−/−) T cells showed a decreasedactivation of these transcription factors compared to their controllittermates (FIG. 5B).

Example 4 Externalization of FPRL-1 and Anx-A1 in T Cells

We investigated whether T cells express the receptor for Anx-A1, theFormyl Peptide Receptor Like-1 (FPRL-1). FACS staining of unstimulatedhuman Peripheral Blood T (PBT) cells with specific monoclonalanti-FPRL-1 antibody demonstrated no receptor expression. However,stimulation with anti-CD3/CD28 induced the externalization of FPRL-1within 1 hour followed by a stable steady state expression on the cellsurface (FIG. 6A). Interestingly, a similar pattern was observed forAnx-A1. Thus, analysis of Anx-A1 distribution in human PBT demonstratedthat the protein is evenly distributed between the cytosol and membrane.However, when cells were stimulated with anti-CD3/CD28, accumulation ofAnx-A1 at the membrane was observed.

The protein is then exported to the outer side of the membrane andreleased into the extracellular milieu. Consistent with this model, whenwe immunoprecipitated Anx-A1 from the culture supernatant of human PBTstimulated with anti-CD3/CD28, we observed an increased release ofAnx-A1 compared to control unstimulated cells (FIG. 6B). Collectively,these observations demonstrate that signalling through the TCR increasesAnx-A1 release, concomitant with the upregulation of its receptors.

In physiological conditions, the Anx-A1/FPRL-1 integrates with the TCRto modulate the strength of TCR signalling. However, in pathologicalconditions, such as in RA or systemic lupus erythematosus (unpublisheddata), where the protein is expressed at higher levels this could leadto increased T cell activation due to lower threshold of TCR signalling(FIG. 6C).

Example 5 Exogenous and Endogenous Anx-A1 modulates Th1/Th2Differentiation

Recent studies have postulated that the strength of TCR signallinginfluences T cell lineage commitment to Th1 or Th2 effector cells. Giventhe increased or decreased TCR signalling in T cells treated withhrAnx-A1 (FIGS. 3 and 5A) or Anx-A1^(−/−) cells (FIGS. 4 and 5B), wethen sought to determine whether different levels of Anx-A1 wouldinfluence T cell differentiation into Th1 or Th2 cells.

Naïve lymph node T cells were differentiated in vitro in Th1 (blackbars) or Th2 (white bars) conditions in presence or absence of hrAnx-A1(600 nM) and then restimulated with platebound anti-CD3 (5.0 mg/ml) for8 h to measure Th1 or Th2 cytokine production. The results are shown inFIG. 7A. Values are mean±S.E. of n=4-5 mice. **P<0.01

As shown in FIG. 7A, differentiation of nave T cells (CD441o, CD62Lhi)in Th1 (anti-CD3/CD28, IL2, IL12 and anti-IL4) or Th2 (anti-CD3/CD28,IL2, IL4 and anti-IFNγ) conditions in the presence of hrAnx-A1 increasedIL2 and IFNγ production with a concomitant decrease of IL4 and IL10release upon anti-CD3 re-stimulation.

Naïve lymph node T cells from Anx-A1^(+/+) or Anx-A1^(−/−) mice weredifferentiated in vitro in Th1 (first and second graphs from the left)or Th2 (third and fourth graphs from the left) conditions and thenrestimulated with platebound anti-CD3 (5.0 mg/ml) for 8 h to measure Th1or Th2 cytokine production. The results are shown in FIG. 7B. Values aremean±S.E. of n=4-5 mice. **P<0.01

As shown in FIG. 7B, similar findings were also obtained with respect tothe endogenous protein: analysis of Th1/Th2 cytokine production indifferentiated Th1/Th2 cells from Anx-A1^(+/+) or Anx-A1⁴⁻ mice yieldedhigher levels of IL2 and IFNγ wild-type mice compared with knockoutmice, with opposite profiles for IL4 and IL13 production.

Example 6 Anx-A1 and Rheumatoid Arthritis

To prove that hrAnx-A1 increased T cell activation in vivo, we used amouse model of chronic autoimmune disease, the collagen-inducedarthritis (CIA) model in DBA mice. Mice were injected with hrAnx-A1daily for 12 days after immunization with collagen (time during whichnave cells differentiate in Th effector cells) and thereafter theprogression of the disease upon antigen challenge was analyzed. FIG. 8shows paw volume (FIG. 8A) and clinical score (FIG. 8B) of the micetreated with PBS (100 μl) or hrAnx-A1 (1 pg s.c. twice a day).Synchronization of disease onset was obtained by boosting with collagenon day 21, and clinical signs were evident from day 22 (day 1 of theonset of the diseases). Values are mean±S.E. of n=6-8 mice. Groups werecompared using the Mann-Whitney test. *P<0.01

As can be seen from FIGS. 8A and 8B, treatment of mice with hrAnx-A1exacerbated the signs and symptoms of arthritis compared to mice treatedwith PBS vehicle, confirming that high levels of Anx-A1 influence T cellactivation and differentiation and that these effects influence thedisease development in a mouse model of RA.

To investigate the clinical relevance of these studies Anx-A1 expressionwas analyzed in CD4+ peripheral T cells and synovial CD3+ cells from RApatients. FIG. 8C shows the results. The median values are indicated byhorizontal lines and p values of the Mann-Whitney test areshown.*P<0.01. As shown in FIG. 8C, RA CD4+ cells express high levels ofAnx-A1 mRNA and protein (data not shown) compared with cells fromhealthy control volunteers (HC).

Fluorescence immunohistochemistry was also carried out using green andred fluorescent tagged secondary antiserum, as shown in each panel ofFIG. 8D. This immunohistochemical analysis of Anx-A1 expression in thesynovial tissue of RA patients revealed a high degree of colocalizationwith CD3+ cells. Therefore, considering that CD4 cells from RA patientsexpress higher levels of Anx-A1, it can be concluded that thedysregulated expression of this protein might contribute to thedevelopment of this disease.

Example 7 Effects of Full Length hrAnx-A1 and the N-Terminal Peptide Ac2-26 on T Cell Activation

The effects of an N-terminal peptide of hrAnx-A1 (peptide Ac 2-26) andof full length hrAnx-A1 on T cell activation were investigated. IL-2production from murine nave lymph node T cells was stimulated with 0.6,1.25 or 2.5 μg/ml of anti-CD3/CD28 in the presence or absence of fulllength hrAnx-A1 (300nM) or the Anx-A1 derived N-terminal peptide Ac.2-26(100pM) for 24 hrs.

It was found that the N-terminal peptide Ac.2-26 retains most of thebiological activity of the full-length protein, i.e. increased IL-2production (FIG. 9) and T cell proliferation (data not shown).

Example 8 Anx-A1 and Atherosclerosis

To investigate if Anx-A1 is expressed in human atherosclerotic plaques,sections of carotid atherosclerotic plaques removed from patients duringcarotid endarteretomy surgery were stained with a mouse monoclonal antihuman Anx-A1 antibody (mAb 1B). The production of this antibody isdescribed in Pepinsky et al FEBS Letters 261: 247-252, 1990. Briefly,BALB/c mice were immunized with an intraperitoneal injection ofannexin-1 (referred to as lipocortin-1 in Pepinsky et al) in completeFreund's adjuvant. The animals were boosted on days 14 and 28 withannexin-1 in incomplete Freund's adjuvant. After 6 weeks, test bleedswere taken and screened for antibodies that blocked annexin-1 activity.Spleen cells from mice whose antisera displayed anti-annexin activitywere fused with SP3×Ag8 cells for hybridoma production. Hybridomaculture supernatants were assayed for antibodies that could precipitateradiolabeled annexin-1, and hybridomas producing antibodies thatprecipitated over 50% of the input counts were subcloned by limitingdilution. The most promising lines were grown as ascites inpristane-primed mice and the monoclonal antibodies wereaffinity-purified on protein A sepharose, using the Pierce binding andelution buffer systems.

As shown in FIG. 10, a compact and clear staining for Anx-A1 could beobserved within the plaque confirming that the inflammatory infiltratewithin these tissues expresses high levels of Anx-A1.

Similar analysis was also carried out in ApoE^(−/−) mice. Localizationof Anx-A1 in the aortic sinus and the brachiocefalic artery (BCA) of 10month old ApoE^(−/−) mice were performed by confocal microscopy todetermine the expression and spatial distribution of Anx-A1. Nonatherosclerotic arterial tissue lacked immunoreactive Anx-A1 (data notshown). In contrast, atherosclerotic plaque from both aortic sinus andBCA stain strongly for Anx-A1 (FIG. 11).

A clear immunoreactivity for Anx-A1 was detected in proximity of thefibrous cap in both aortic sinus (FIG. 11A) and BCA (FIG. 11B) and inproximity of the necrotic core of the plaque in the aortic sinus (FIG.11B). These results demonstrate that Anx-A1 is expressed in both humanand murine atherosclerotic plaques and suggest that its expression couldpotentially influence plaque stability.

Example 9 Anx-A1 and Systemic Lupus Erythematosus (SLE)

Clinical studies on the biological functions of Annexin-1 haveassociated the presence of autoantibodies against this protein with thedevelopment of autoimmune diseases including systemic lupuserythemathosus (SLE), rheumatoid arthritis and inflammatory boweldisease. In light of these findings we hypothesized that the generationof these autoantibodies might be due to an uncontrolled expression ofAnnexin-1 in these patients. To verify this hypothesis, the expressionlevel of Annexin-1 in T cells collected from healthy volunteers and SLEpatients was analyzed. Annexin-1 mRNA and protein were expressed at amuch more marked level in the SLE T cells (FIG. 12). Thus, these resultssupport the hypothesis that increased Annexin-1 expression in SLE Tcells, and therefore in T cells from patients with other autoimmunepathologies, might be responsible for the increased levels of Th1cytokines described in these pathologies, thereby representing a riskfactor for the development of autoimmune diseases.

Example 10 Inhibition of T Cell Activation by Anti-Anx-A1 Antibodies

Purified human peripheral blood T cells were incubated with a mixture ofanti-CD3 and anti-CD28 antibodies (5 mg/ml) to activate the T cellreceptor (TCR): this occurred as demonstrated in FIG. 13 by theremarkable production of interleukin-2 (IL-2), a cytokine central to Tcell activation and differentiation.

Cells were then incubated with different concentrations (1.0, 0.1, 0.01and 0.001 micrograms/ml) of a neutralising mouse monoclonal antibodyraised against human recombinant annexin 1 (mAb 1A). The production ofthis antibody is described in Pepinsky et al FEBS Letters 261: 247-252,1990 and in Example 8.

Treatment with mAblA produced a concentration dependent inhibition ofIL-2 production (FIG. 13) and cell proliferation (data not shown). IgGwas used as a control at concentrations of 1.0, 0.1, 0.01 and 0.001micrograms/ml and was without efficacy at all concentrations. Theresults showed that blockade of annexin-1 effects seemed to be moreeffective at lower concentrations of the specific monoclonal antibodymAblA.

In all cases, data are mean±SE of triplicate measurements. *P<0.01.

Purified human peripheral blood T cells from a different donor were thenincubated with a mixture of anti-CD3 and anti-CD28 antibodies at adifferent concentration (1 mg/ml) to activate the T cell receptor (TCR).Again, this occurred as demonstrated in FIG. 14 by the production ofinterleukin-2 (IL-2), but at a lower level due to the lowerconcentration of anti-CD3 and anti-CD28 antibodies used.

Again, cells were then incubated with different concentrations (1.0,0.1, 0.01 and 0.001 micrograms/ml) of the neutralising mouse monoclonalantibody raised against human recombinant annexin 1 (mAb 1A). Treatmentwith mAblA produced a concentration dependent inhibition of IL-2production (FIG. 14). IgG was used as a control at a concentration of1.0 micrograms/ml and was without efficacy. Again, the results showedthat blockade of annexin-1 effects seemed to be more effective at lowerconcentrations of the specific monoclonal antibody mAb 1A, except with aconcentration of 1.0 micrograms/ml of mAblA.

In all cases, data are mean±SE of triplicate measurements. *P<0.01.

These experiments demonstrate that endogenous annexin 1 promotes T cellactivation in the presence of a specific stimulus. In addition, blockadeof the annexin 1 pathway attenuated T cell activation by up to 50%. Thefact that inhibition reached a maximum of around 50% suggests that“normal and housekeeping” immunity would not be affected by treatment ofT cell-mediated diseases using a molecule which specifically binds toAnx-A1, as claimed.

Example 11 Anx-A1 and Multiple Sclerosis (MS)

Materials and Methods

Reagents

The Myelin Oligodendrocyte Glycoprotein peptide (MOG)₃₃₋₅₅(MEVGWYRSPFSRVVHLYRNGK) was synthesized and purified by CambridgeResearch Biochemicals (Billingham, UK). Complete Freund's adjuvantcontaining Mycobacterium tuberculosis H37a was purchased from Difcowhile Bordetella pertussis toxin was from Sigma-Aldrich Co (Poole, UK).Unless otherwise specified, all the other reagents were fromSigma-Aldrich Co.

Mice

Male AnxA1 null mice were as previously described (Hannon et al., FasebJ, 17: 253-255, 2003; Roviezzo et al., J. Physiol Pharmacol 53: 541-553,2002) (9-11 week old) and were backcrossed on a C57BL/6 backgroundfor >10 generations and bred at

B&K animal care facilities (Hull, UK). Age and gender-matched controlC57BL/6 mice were used as control for all experiments. Animals were keptunder standard conditions and maintained in a 12h/12h light/dark cycleat 22 ±1° C. in accordance with United Kingdom Home Office regulations(Animal Act 1986) and of the European Union directives.

Induction of EAE

Mice were immunized subcutaneously on day 0 with 300 μl of emulsionconsisting of 300 pg of MOG₃₅₋₅₅ in PBS combined with an equal volume ofCFA containing 300 μg heat-killed M. tuberculosis H37Ra. The emulsionwas injected in both flanks and followed by an intraperitoneal injectionof B. pertussis toxin (500 ng/100 μl) in 100 μl of saline on days 0 and2. Mice were observed daily for signs of EAE and weight loss. Diseaseseverity was scored on a 6-point scale: 0=no disease; 1=partial flaccidtail; 2=complete flaccid tail; 3=hind limb hypotonia; 4=partial hindlimb paralysis; 5=complete hind limb paralysis; 6=moribund or deadanimal

Cell Proliferation Assay

Lymph node cells (10⁵ cells/200 μl) obtained from mice immunized withMOG₃₃₋₅₅ for 14 days were stimulated with MOG₃₃₋₅₅ (50-100 μg/200 μl)for 48 h in 96 well plates. During the last 12 h, cultures were pulsedwith 1 μCi of [³H]-thymidine (Amersham Pharmacia Biotech,Buckinghamshire, UK) and incorporated radioactivity was measured byautomated scintillation counter (Packard Instrument Company, Inc., Ill.,US).

Cytokine ELISA

Lymph node cells (10⁶ cells/ml) obtained from mice immunized withMOG₃₃₋₅₅ for 14 days were stimulated with MOG₃₃₋₅₅ (100 μg/ml) for 4days. Cell supernatants were collected and analyzed for IFN-γ, IL-2,IL-17A and TNF-α content using ELISA kits (eBioscience, Dorset, UK)according to manufacturer's instructions.

Isolation of Inflammatory Cell from the Spinal Cord

Mice were killed using CO₂. The spinal cords were expelled from thespinal column with PBS by hydrostatic pressure using a syringe attachedto a 21-gauge needle.

Tissues were cut in small pieces and passed through cell strainer (70nm; BD Falcon) using the plunger of a sterile 1 ml syringe. The singlecell suspension was centrifuged for 10 min at 390×g, resuspended in 20ml of PBS containing 30% of Percoll (Sigma) and overlayed onto 10 ml ofPBS containing 70% Percoll. After centrifugation at 390xg for 20 min,the mononuclear cells were removed from the interphase, washed, andresuspended in FACS buffer (PBS containing 1% FCS and 0.02% NaN₂) forfurther analysis.

Flow Cytometry

Cell samples from Percoll-purified spinal cord tissues orFicoll-purified lymph nodes were resuspended in FACS buffer containingCD16/CD32 FcyIIR blocking antibody (clone 93; eBioscience) for 30 min at4° C. Thereafter, cell suspensions were labelled with theFITC-conjugated anti-CD3 (1:100; clone 145 2C11) or anti-F4/80 (1:100;clone BMT) while lymph node cells were stained with anti-CD4-FITC(1:500; clone L3T4) and anti-CD8 (1:1000; clone Ly-2) for 30 min at 4°C., prior to analysis by FACS calibur using CellQuest software (BectonDickinson). At least 10⁴ cells were analyzed per sample, anddetermination of positive and negative populations was performed basedon the staining attained with irrelevant IgG isotypes.

Histology

Spinal cord tissues were dissected and fixed in 4% neutral bufferedformalin for 48 hrs and then incubated with decalcifying solutioncontaining EDTA (0.1 mM in PBS) for 14 days prior to paraffin embedding.Histological evaluation was performed on paraffin-embedded sectionssampled at various time points depending on disease severity. Spinalcord sections (5 μm) were deparaffinized with xylene and stained withhaematoxylin and eosin (H&E) to assess inflammation. The staining forAnxA1 was performed on frozen sections using anti-AnxA1 (dilution 1:500;Zymed, Invitrogen) and anti-rabbit Ig horseradish peroxidase(HRP)-conjugated antibodies (dilution 1:500; Dako). Double staining forAnxA1 and CD3 or F4/80 was carried out as previously described usingFITC-conjugated anti-CD3 (1:100; clone 145 2C11) or anti-F4/80 (1:100;clone BMT). Sections were also counterstained with haematoxylin.

In all cases, a minimum >3 sections per animal were evaluated.Phase-contrast digital images were taken using the Image Pro imageanalysis software package.

Statistical Analysis

Prism software (GraphPad software) was used to run all the tests.Statistical evaluations of cell frequency, proliferation and cytokineproduction were performed using two-tailed, unpaired Student's t tests.ANOVA were applied to analyze the EAE clinical grading. A p value of<0.05 was considered to be statistically significant. P-values lowerthan 0.05 were considered significant. Data are presented as mean±S.E.Mof n samples per group.

Results

AnxA1 expression correlates with the severity of experimental autoimmuneencephalomyelitis (EAE)

The correlation between AnxA1 levels in the spinal cord content andextent of infiltrating mononuclear cells in the CNS were assessed in amouse model of MS induced by immunization with MOG₃₅₋₅₅, EAE.MOG₃₅₋₅₅-induced EAE is a model for autoimmune demyelination of the CNSand has been widely used to investigate pathogenic mechanismsresponsible for the development of MS. To this aim, spinal cords andbrains of wild type mice immunized with MOG₃₅₋₅₅ peptide at differentstages of the diseases i.e. at day 12 (score 0), day 18 (score 2) andday 20 (score 4) were collected and immunohistochemistry for AnxA1 wasperformed side by side with hematoxylin and eosin staining

As shown in FIG. 15, spinal cord tissues collected during the inductionphase of mice with no signs of disease showed a faint staining for AnxA1(score 0, FIGS. 15A and B, respectively). However, with the onset ofclinical signs and the appearance of inflammatory infiltrates in theCNS, discrete patches of AnxA1 immunostaining were observed all aroundthe meninges (score 2, FIG. 15A and B, respectively). As the diseaseprogressed, an increase in number of AnxA1-positive cellular infiltratepatches was observed (score 4, FIG. 15A and B, respectively), suggestingthat the infiltration of inflammatory cells expressing high levels ofAnxA1 is correlated with the severity of the disease.

To identify the cellular sources of AnxA1 immunoreactivity in the spinalcord, double immunofluorescence staining of the sections was performedwith anti-AnxA1 and either anti-CD3 (marker for T cells) or anti-F4/80(marker for macrophages). A large number of infiltrated T cells andmacrophages was detected in the spinal cord sections of mice at the peakof EAE (FIG. 16A and B, middle panels, respectively). However, AnxA1staining in the same sections showed a partial co-localization with bothT cells and macrophages without particular preference for one or theother cell types (FIG. 16A and B, right panels, respectively).

AnxA1^(−/−) Mice Develop an Impaired EAE

Since AnxA1 expression was upregulated at the peak of EAE, the role ofthis protein on the development of EAE was investigated. AnxA1^(+/+) andAnxA1^(−/−) mice were immunized s.c. with MOG₃₅₋₅₅ peptide in CFA on day0, and then injected i.v. with B. pertussis toxin on both day 0 and day2. Both AnxA1^(+/+) and AnxA1^(−/−) mice started to develop EAE from day12 after immunization, reaching peak disease around day 20. However,AnxA1^(−/−) mice had reduced levels of disease compared to AnxA1^(+/+)(FIG. 17A). Interestingly, this was evident and significant only at thelater stage of the disease i.e. from day 18 to 23 and onwards.

Studies on animal models of EAE have demonstrated that the acute phaseof the disease coincides with weight loss, probably due to anorexia anddeficient fluid uptake. Weight measurement of immunized mice correlatedwith the severity of the clinical score and showed a reduced weight lossfrom day 18 onwards—in the AnxA1^(−/−) mice compared to AnxA1^(+/+)controls (FIG. 17B). Further comparison of development of EAE inAnxA1^(+/+) and AnxA1^(−/−) mice showed a decrease in both the mortalityrate and maximum disease score, without differences in the incidencerate or disease onset (Table 1).

TABLE 1 Clinical parameters of MOG₃₅₋₅₅-induced EAE in AnxA1^(+/+) andAnxA1^(−/−) mice (mean ± SEM, n = 10/group) Onset day Max. score MiceIncidence^(§) Mortality (mean ± SEM) (mean ± SEM) AnxA1^(+/+) 100% 33.3%16.4 ± 2.3 5.7 ± 0.2  (10/10) (3/10)  AnxA1^(−/−) 100%   0% 15.9 ± 1.34.3 ± 0.1** (10/10) (0/10)** **p < 0.01, representative of 3 experiments^(§)EAE clinical score equal or greater than 1.

In vitro Recall Response to MOG₃₅₋₅₅ in AnxA1^(−/−) Mice

T cells play a key role in the development of EAE and AnxA1^(−/−) Tcells have an impaired capacity to respond to anti-CD3/CD28 stimulation.In light of these findings, it was investigated whether the decreaseddevelopment of EAE in AnxA1^(−/−) mice was associated with a lowerresponse to antigen-stimulation. Lymph node cells from AnxA1^(+/+) andAm(A1^(−/−) mice, collected 14 days after immunization, were stimulatedin vitro with MOG₃₅₋₅₅. ArD(A1^(−/−) lymph node cells showed a decreasedrate of proliferation and produced lower levels of IL-2 when stimulatedwith MOG₃₅₋₅₅ compared to wild-type mice (FIGS. 18A and B,respectively). Similar results were obtained with splenocytes (data notshown).

These results on cell proliferation were mirrored in the number of cellsrecovered from the spleen and the draining lymph nodes of the immunizedmice. The total cell count of Ficoll-purified spleen and lymph nodemononuclear cells from the same animals, revealed a significant decreasein AnxA1^(−/−) mice compared to controls (FIG. 19A and B, respectively),with no measurable changes in the percentages of CD4 or CD8 positivecells (FIG. 19C and D, respectively).

Reduced MOG₃₅₋₅₅-Specific Th1 and Th17 Cytokine Responses in AnxA1^(−/−)Mice

Studies using draining lymph node cells from MOG₃₅₋₅₅ immunized C57/BL6mice showed significant changes in Th1 and Th17 cytokine production.Analysis of cytokine production from AnxA1^(−/−) lymph node cells uponre-challenge with MOG₃₅₋₅₅ for 96 h showed a decreased production of Th1cytokines IFN-γ, IL-2, and TNF-α compared to wild type cells (FIG.20A-C). Similarly, measurement of Th17 signature product IL-17, revealeddecreased levels of this cytokine in AnxA1^(−/−) compared to wild type(FIG. 20D).

T cell infiltration in the nervous system of AnxA1^(−/−) mice during EAEThe reduced signs of EAE in AnxA1^(−/−) mice from day 18 onwards,prompted us to investigate whether there could be a neuro-pathologicalcorrelate. The spinal cords of AnxA1^(+/+) and AnxA1^(−/−) treated mice,collected at day 18 or 22, were analyzed for histological evidence ofinflammation. It was found that there were reduced numbers of immunecell infiltrates detected in AnxA1^(−/−) mice compared to AnxA1^(+/+)animals. (FIG. 21A and B).

The reduced histological signs of inflammation in AnxA1^(−/−) mice wereassociated with a reduced number of CD3 and F4/80 positive cellsinfiltrating the CNS (FIG. 21C and D, respectively). These qualitativeanalyses were confirmed by FACS measuring the percentages of CD3 andF4/80 positive leucocytes isolated from day 18 spinal cord tissues.Consistent with the immunohistochemistry results, Anx1^(−/−) mice hadabout 60 and 80% less T cells and macrophages, respectively, compared toAnxA1^(+/+) mice (FIG. 22A and B, respectively).

The results show that there is a remarkable accumulation of Annexin-1expressing cells in the spinal cord of mice at the peak of the disease.There is therefore a correlation between Annexin-1 expression and thedevelopment of EAE.

In addition, the results show that Annexin-1 deficient mice develop lesssevere EAE. Ablating Annexin-1 expression therefore limits thedevelopment of EAE, a mouse model for MS.

1-12. (canceled)
 13. A method of treating a T cell-mediated disease inan animal comprising administering to the animal in need thereof atherapeutic amount of an antibody, or fragment thereof, that binds toAnnexin-1 (Anx-A1).
 14. The method of claim 13 wherein the antibodybinds to an N-terminal peptide of Anx-A1 of at least 50 amino acidresidues.
 15. The method of claim 13 wherein the antibody binds to theN-terminal peptide Ac.2-26 of Anx-A1.
 16. The method of claim 13 whereinthe antibody binds to a fragment of at least 6 amino acids of theN-terminal peptide Ac.2-26 of Anx-A1.
 17. The method of claim 13 whereinthe antibody is a monoclonal antibody.
 18. The method of claim 13wherein the antibody fragment is chosen from Fab fragment, F(ab')₂fragment, Fv fragment, and single chain Fv (scFv) molecule.
 19. Themethod of claim 13 wherein the antibody is a chimeric antibody.
 20. Themethod of claim 13 wherein the antibody is a humanized antibody.
 21. Themethod of claim 13 wherein the T cell-mediated disease is chosen fromgraft-versus-host disease, graft rejection, atherosclerosis, HIV, AIDS,psoriasis, and an autoimmune disease.
 22. The method of claim 13 whereinthe T cell-mediated disease is chosen from atherosclerosis, HIV, AIDS,and psoriasis.
 23. The method of claim 21 wherein the autoimmune diseaseis chosen from rheumatoid arthritis, multiple sclerosis, systemic lupuserythematosus, Addison's disease, Grave's disease, scleroderma,polymyositis, diabetes mellitus, autoimmune uveoretinitis, ulcerativecolitis, pemphigus vulgaris, inflammatory bowel disease, and autoimmunethyroiditis.
 24. The method of claim 21 wherein the autoimmune diseaseis chosen from rheumatoid arthritis, multiple sclerosis, and systemiclupus erythematosus.